IMPA2 blocks cervical cancer cell apoptosis and induces paclitaxel resistance through p53-mediated AIFM2 regulation

Cervical cancer continues to be a concern, and the prognosis of locally advanced cervical cancer remains poor. IMPA2 was previously identified as a potential oncogene and regulator of tumor apoptosis. In this study, we aim to further elucidate the underlying mechanisms of IMPA2 gene in the regulation of cervical cancer apoptosis. First, we identify AIFM2 as an upregulated gene in IMPA2-silenced cervical cancer cells, and inhibition of AIFM2 reverses IMPA2 knockdown-induced apoptosis. Further study reveals that AIFM2 regulates cell apoptosis in a mitochondrial-dependent manner with a redistribution of mitochondrial membrane potential and intracellular Ca 2+ levels. However, the analysis of the STRING database and our experimental results show that AIFM2 has little effect on cervical cancer progression and survival. Further mechanistic study demonstrates that IMPA2 and AIFM2 silencing inhibits apoptosis by activating p53. Meanwhile, the knockdown of IMPA2 enhances the chemosensitivity of cervical cancer cells by strengthening paclitaxel-induced apoptosis. Based on the above results, the IMPA2/AIFM2/p53 pathway may be a new molecular mechanism for paclitaxel treatment of cervical cancer and an effective strategy to enhance the sensitivity of cervical cancer cells to paclitaxel. Our findings display a novel function of IMPA2 in regulating cell apoptosis and paclitaxel resistance mediated by a disturbance of AIFM2 and p53 expression, potentially making it a novel therapeutic target for cervical cancer treatment.


Introduction
Cervical cancer is a common health problem that seriously threatens women's health worldwide, with approximately 12,820 new cases and 4210 deaths per year in the US [1,2]. Radiotherapy and chemotherapy are still the two main treatments for cervical cancer. However, the prognosis of locally advanced cervical cancer remains poor, and treatment still results in substantial morbidity due to chemotherapy drug resistance [3,4]. Therefore, alternative therapeutic strategies are needed. Recently, the clinical potential of activators of apoptotic pathways has been widely studied in treating cervical cancer [5]. Thus, better knowledge of the molecular mechanisms modulating apoptotic pathways is urgently needed.
Most studies on IMPA2 have focused on neuropsychiatric diseases and the pharmacological action of lithium [7,8]. It was reported that altered IMPA2 gene expression was closely related to calcium homeostasis in bipolar disorder [9]. Recently, Lin et al. [10] found that dysregulation of IMPA2 could promote metastatic progression of clear cell renal cell carcinoma. Our previous study demonstrated that IMPA2 could play a tumor-promoting role in cervical cancer for the first time, and proteomic analysis and flow cytometry analysis of apoptosis results showed that IMPA2 may regulate the apoptosis process of cervical cancer [11], but the underlying mechanisms are still unknown. Apoptosis-inducing factor mitochondria associated 2 (AIFM2/ FSP1) was found to be upregulated in our previous proteomics analysis when IMPA2 gene expression was silenced in cervical cancer cells. It was reported that AIFM2 could play a role in mitochondrial stress signaling and enhance apoptosis of human lung cancer cells [12]. Tan et al. [13] also proved that ATF6 could aggravate acinar cell apoptosis and injury by regulating p53/AIFM2 transcription in severe acute pancreatitis. Although evidence has shown that AIFM2 may contribute to cell apoptosis, little research has focused on its role in cervical cancer. Our previous study first showed that IMPA2 might promote cervical cancer progression [11].
In this study, we revealed that silencing of IMPA2 could aggravate apoptosis by activating the p53 signaling pathway and upregulating the expression of AIFM2 and induce paclitaxel resistance through the IMPA2/AIFM2/P53 pathway. Our results suggest that IMPA2 is a potential therapeutic target for cervical cancer treatment.

Tissue samples
Tissue samples for Bak and AIFM2 immunohistochemistry (IHC) staining were xenografts from mice that were injected with IMPA2silenced or control SiHa cells derived from our previous study [11]. Tissue samples for AIFM2 mRNA detection and IHC staining were obtained from three patients with written consents who underwent radical hysterectomy at the Second Xiangya Hospital, Central South University. This study was approved by the Joint Ethics Committee of the Central South University Health Authority (No. 2020046, Date: 2020/04/01) and performed in accordance with the Declaration of Helsinki.

Flow cytometric analysis
After transfection for 6 h, the cells were incubated for 48 h at 37°C, in a 5% CO 2 incubator. The collected cells were then rinsed twice with PBS and prepared for subsequent testing. After treating IMPA2-silenced SiHa and HeLa cells with 0 μM and 20 μM PFT-α (Sigma) for 24 h, apoptosis was detected using an Annexin-V-APC/PI staining kit (Biolegend, Shanghai, China) according to the manufacturer's instructions and finally analysed on a Guava easyCyte HT flow cytometer (Millipore, Billerica, USA).

Measurement of the intracellular Ca 2+ concentration
The mitochondrial Ca 2+ concentration was detected using a Rhod-2 AM probe (Maokang, Shanghai, China). Detection was carried out according to the manufacturer's instructions, immunofluorescence signals were examined using the A1 fluorescence microscope, and ImageJ was used for fluorescence quantification.

Measurement of mitochondrial membrane potential
The extent of mitochondrial membrane potential (MMP) loss was measured using the potentiometric cation JC-1 (MedChemExpress). Transfected HeLa or SiHa cells were incubated with JC-1 staining solution for 20 min at 37°C and examined under the A1 fluorescence microscope.

CCK-8 cell viability assay
Cells transfected with siAIFM2 or siNC were seeded into a 96-well plate at 2×10 3 cells per well with 100 μL of culture medium and cultured for 24, 48, 72, and 96 h at 37°C and 5% CO 2 . Cell viability was determined using a CCK8 assay kit (KeyGen Biotech, Nanjing, China) according to the manufacturer's instructions. Each process was repeated three times.

RNA isolation and quantitative real-time PCR
Total RNA was extracted using TRIzol reagent (Sangon Biotech). RNA (1 μg) was reverse transcribed into cDNA using the Transcriptor First Strand cDNA Synthesis Kit (Roche Diagnostics, Mannheim, Germany) according to the supplier's instructions. Quantitative real-time PCR analysis was performed with a Stratagene Mx3000P qPCR system (Agilent Technologies, Santa Clara, USA) using Thunderbird qPCR Mix (Toyobo, Osaka, Japan).

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IMPA2 blocks cervical cancer cell apoptosis cDNA samples were tested in triplicate, and glyceraldehyde-3phosphate dehydrogenase (GAPDH) was used as a reference gene. The expression of genes was quantified by measuring Ct values and normalized using the 2 -ΔΔCt method relative to GAPDH. The primer pairs used for qRT-PCR were designed using the Primer3 program.
The primers used are shown in Table 1.

Western blot analysis
The cell extracts were prepared using RIPA buffer (KeyGen Biotech) containing protease inhibitors (KeyGen Biotech). Equal amounts of protein samples were subjected to 12% SDS-PAGE and transferred to PVDF membranes (Immobilon-P; Millipore, Billerica, USA). The membranes were then blotted with primary antibodies overnight at 4°C, followed by incubation with the HRP-conjugated goat antirabbit IgG secondary antibody (1:5000). The antibodies used were as follows: anti-AIFM2 ( The protein bands were then visualized using enhanced chemiluminescence reagent (Bio-Rad, Hercules, USA). Band quantification was conducted using ImageJ (National Institutes of Health, Bethesda, USA).

Statistical analysis
Data are shown as the mean±standard deviation (SD) based on at least three independent experiments. The results were analysed using SPSS 22.0 (SPSS, Chicago, USA) and GraphPad Prism 6 software (GraphPad, San Diego, USA). Two-tailed Student's t-test was used to evaluate the difference between two data groups. A P value<0.05 was considered statistically significant.

IMPA2 knockdown upregulates AIFM2 gene expression to induce apoptosis of HeLa and SiHa cells
To investigate the underlying mechanisms of the modulation of IMPA2 in the apoptotic process of cervical cancer cells, we screened 8 molecules related to apoptosis from the previous proteomic results. We created the STRING database (http://string-db.org) to find the interacting molecules ( Figure 1A). After silencing IMPA2, only AIFM2 mRNA expression was upregulated in HeLa and SiHa cells ( Figure 1B). Similar results were obtained in the protein expression level detected by western blot analysis ( Figure 1C). In addition, immunofluorescence results also showed that IMPA2 knockdown increased AIFM2 expression in both HeLa and SiHa cells ( Figure 1D,E). Meanwhile, IHC staining also demonstrated higher AIFM2 protein expression in xenografts from mice after injecting shIMPA2-Siha cells (P<0.05; Figure 1F,G). These results indicated that inhibition of IMPA2 expression could upregulate AIFM2 gene expression. Furthermore, IMPA2 expression knockdown-induced changes in apoptosis-related proteins were reversed by cotransfecting with AIFM2 siRNA, with an increase in Bcl-2 protein level and decreases in Bax and Caspase3 protein levels ( Figure 1H-J). Similarly, apoptotic cells were also decreased when AIFM2 and IMPA2 genes were simultaneously knocked down in cervical cancer cells ( Figure 1K-M). All these data suggested that IMPA2 inhibition could promote apoptosis of HeLa and SiHa cells by activating AIFM2 expression.

Downregulation of AIFM2 represses mitochondriadependent apoptosis
Some articles have proven the crucial role of AIFM2 in apoptosis, but few studies have focused on its function in cancer cells, including cervical cancer cells. In the above section, we found that AIFM2 could be upregulated in IMPA2-silenced HeLa and SiHa cells. To further explore whether AIFM2 affects apoptosis in cervical cancer cells, we designed three siRNAs to knockdown AIFM2 gene expression. As shown in Figure 2A,B, AIFM2 was significantly downregulated at both the mRNA and protein levels (P<0.01). Among the three siRNAs, si-1 had the most significant inhibitory effect on HeLa cells, and si-2 suppressed most SiHa cells. Based on this, we chose si-1 and si-2 for our subsequent experiments. As shown in Figure 2C,D, AIFM2-knockdown cells had lower expression of Bax and Caspase3, consistent with an increase in Bcl-2 expression. Meanwhile, the percentage of apoptotic cancer cells transfected with AIFM2 siRNA was also decreased compared with that in the control group ( Figure 2E-G).
On the other hand, in AIFM2-knockdown HeLa and SiHa cells, the intensity of green fluorescence was weaker, and the red fluorescence was stronger after JC-1 dye staining, while opposite results were obtained in the control cells. These results indicated a redistribution of the mitochondrial membrane potential ( Figure  2H,I). In addition, AIFM2-knockdown cells also had a lower concentration of mitochondrial Ca 2+ (P<0.001). These results demonstrated that AIFM2 could regulate the apoptosis of cervical

AIFM2 inhibition has little effect on cervical cancer progression and survival
To explore the function of AIFM2 in tumors, AIFM2 expression was first queried in the GCBI database (http://college.gcbi.com.cn/) (Supplementary Figure S2A). Then, the analysis of a public CESC (Cervical squamous cell carcinoma) dataset from The Cancer Genome Atlas (TCGA) (https://tcga-data.nci.nih.gov/) showed that there was no significant difference in AIFM2 expression between cancer tissues and normal tissues (Supplementary Figure  S2B). The survival rates of patients with high AIFM2 expression and patients with low AIFM2 expression were also not obviously different (P=0.55; Supplementary Figure S2C). To confirm the results from the databases, we further detected AIFM2 expression in 4 pairs of cervical cancer tissues and the corresponding paracarcinoma tissues. No significant difference was found in AIFM2 expression between cancer tissues and the corresponding paracarcinoma tissues (Supplementary Figure S2D). Similar to the tissue results, silencing of AIFM2 in SiHa and HeLa cells had little effect on

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IMPA2 blocks cervical cancer cell apoptosis cervical cancer cell growth (Supplementary Figure S2E,F). Additionally, AIFM2 expression in cervical cancer tissues and normal tissues showed no significant difference, as detected by IHC staining (Supplementary Figure S2G). In summary, AIFM2 expression may have little effect on cancer progression and survival, which may be explained by the complex functions of AIFM2, including the induction of apoptosis and suppression of ferroptosis.

IMPA2 knockdown triggers AIFM2-mediated apoptosis via the p53 signaling pathway in HeLa and SiHa cells
To further understand the mechanisms involved in IMPA2-AIFM2regulated cell apoptosis, we identified some classical pathways of apoptosis regulation in cancer cells. PI3K/AKT/mTOR, JAK/STAT3, and p53 proteins were detected by western blot analysis in IMPA2silenced cells and control cells ( Figure 3A). The results showed that mTOR protein was activated, but PI3K and AKT were only activated IMPA2 blocks cervical cancer cell apoptosis 627 in HeLa or SiHa cells, suggesting that PI3K/AKT/mTOR may be partially involved in IMPA2-regulated cell apoptosis. In addition, IMPA2 silencing significantly increased JAK/STAT3 phosphorylation and p53 expression in SiHa and HeLa cells, indicating that IMPA2 may affect apoptosis via the JAK/STAT3 pathway or p53 pathway. Additionally, proteins in apoptotic pathways were detected in the AIFM2-knockdown cells and control cells. As shown in Figure 3B, JAK/STAT3 phosphorylation was repressed, and p53 expression was also decreased after AIFM2 gene inhibition. Furthermore, IMPA2 silencing-induced p53 activation was suppressed by inhibition of AIFM2. At the same time, the phosphorylation of JAK/STAT3 was not rescued by siAIFM2 ( Figure 3C), suggesting that p53 may be a key molecule for IMPA2-AIFM2regulated tumor apoptosis. In addition, the relationship between AIFM2 and p53 was also verified from the STRING database ( Figure  3D).

PFT-α treatment successfully rescues p53-induced cell apoptosis in IMPA2-silenced HeLa and SiHa cells
To further identify the roles of p53 and JAK/STAT3 in cervical cancer apoptosis induced by IMPA2 and AIFM2, we examined the effects of PFT-α and AG490 on apoptosis proteins. As shown in Figure 4A,

IMPA2 enhances paclitaxel resistance by inhibiting apoptosis in cervical cancer cells
Cellular drug resistance is now a challenge in the clinical treatment of cervical cancer. It has been shown that inhibition of apoptosis is one of the leading causes of tumor chemotherapy resistance. We selected the clinical first-line drugs cisplatin and paclitaxel as the study subjects. We found that knockdown of IMPA2 had no significant effect on the tumor cell killing effect of cisplatin after IMPA2 expression was inhibited ( Figure 5A,B). However, there was a significant synergistic effect on paclitaxel. After knockdown of IMPA2, the IC 50 values of paclitaxel were decreased in both HeLa and SiHa cells ( Figure 5C,D), indicating that inhibition of IMPA2 expression could enhance the sensitivity of tumor cells to paclitaxel. Then, different concentrations of paclitaxel were used to treat IMPA2-knockdown cells and negative control cells, and significant differences in apoptosis-related proteins were found in HeLa cells

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IMPA2 blocks cervical cancer cell apoptosis ( Figure 5E) and SiHa cells ( Figure 5F). Treatment with paclitaxel caused a decrease in anti-apoptotic proteins ( Figure 5G) and an increase in pro-apoptotic proteins ( Figure 5H-J) compared with the untreated group. Compared with the control cells, the apoptosis of cells was significantly enhanced in the shIMPA2 group after treatment with paclitaxel, indicating that knockdown of IMPA2 could synergize with the proapoptotic effect of paclitaxel and increase the sensitivity of cervical cancer cells to paclitaxel. Meanwhile, 20 and 40 ng/mL of paclitaxel were also found to significantly inhibit the expression of IMPA2 and upregulate the expressions of AIFM2 and p53 ( Figure 5K-M), suggesting that paclitaxel may exert its tumor-killing effect by regulating the expression of IMPA2/AIFM2/p53 and that IMPA2 may be a new target for the action of paclitaxel. To further demonstrate that IMPA2 can generate drug resistance by affecting paclitaxelmediated apoptosis, we selected cervical cancer cells with stable knockdown of IMPA2 and control cells. We treated them with appropriate concentrations of paclitaxel based on the western blot analysis results. We found that apoptotic cells were increased in cervical cancer cells with IMPA2 knockdown after treatment with paclitaxel compared with that in control cells ( Figure 5N-Q). The paclitaxel-induced apoptosis was concentration-dependent in HeLa cells ( Figure 5N,O). This indicates that inhibition of IMPA2 expression can synergize with paclitaxel-induced apoptosis and thus promote the sensitivity of cervical cancer cells to paclitaxel.

Discussion
Due to an increase in cervical screening and the popularization of the HPV vaccine, deaths from cervical cancer have decreased in recent years. However, the prognosis of cervical cancer remains poor, especially in developing countries [14]. Resistance to chemotherapy drugs contributes to treatment failure or tumor recurrence, and molecular targeted therapy displays a remarkable curative effect. Thus, developing new molecules for cervical cancer screening and treatment is urgently needed. In this study, IMPA2, a novel gene we previously found from transcriptomics analysis [11], was proved to be a potential tumor-promoting gene. We further confirmed that IMPA2 might affect cancer cell death by inducing IMPA2 blocks cervical cancer cell apoptosis 629 cell apoptosis. AIFM2, a NAD(P)H-dependent oxidoreductase involved in the cellular oxidative stress response [15], has also been engaged in IMPA2 knockdown-mediated cell apoptosis by activating p53. This evidence suggests that IMPA2 is a potential target for cervical cancer therapy. IMPA2 irregularity has always been proven to contribute to the pathophysiology of bipolar disorder [6,16,17]. Few studies have focused on its other functions. In this study, we found that IMPA2 could affect the survival of cervical cancer cells by disturbing the apoptotic process. It was reported that altered IMPA2 gene expression is related to calcium homeostasis in bipolar disorder [9], providing indirect evidence of a relationship between IMPA2 and cell apoptosis.
Although we have confirmed the proapoptotic function of AIFM2 in cervical cancer, conflicting data exist on the proapoptotic function of the protein in different diseases [18][19][20]. By comparing the viability of AIFM2-silenced cells and control cells, we found that there was no significant difference (Supplementary Figure S2), indicating that apart from apoptosis, other processes regulated by AIFM2 may also be involved in cervical cancer cell death. It has been reported that FSP1/AIFM2 is a glutathione-independent ferroptosis suppressor, which acts parallel to glutathione peroxidase 4 (GPX4) to inhibit ferroptosis [21,22]. Ferroptosis is a newly discovered form of regulated cell death, the nexus between metabolism and human health [23]. As AIFM2 encodes FSP1, which blocks ferroptosis and induces mitochondrial-dependent cell apoptosis, we did not find apparent changes in cell viability after inhibiting AIFM2 expression. Moreover, to explore whether ferroptosis is involved in IMPA2-regulated cell death, we detected GPX4 expression. The results showed that there is no significant difference between IMPA2-knockdown cells and control cells (Supplementary Figure S1), suggesting that IMPA2-induced cancer

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IMPA2 blocks cervical cancer cell apoptosis progression may depend on ferroptosis. Cisplatin and paclitaxel are the first-line drugs for clinical cervical cancer treatment. With the emergence of clinical drug resistance, the effectiveness of chemotherapeutic drugs decreases. Therefore, exploring the mechanism of drug resistance of chemotherapeutic drugs and enhancing the sensitivity of drugs to cancer cells have become hot spots and focuses of continuous research to improve the survival rate of patients.
IMPA2, as a potential oncogene, was well established in this study to regulate the onset of apoptosis in cervical cancer cells. It has also been found that inhibition of IMPA2 expression can synergize with the tumor-killing effect of paclitaxel. Nevertheless, it had no significant impact on the efficacy of cisplatin ( Figure 5A-D), which is related to the different mechanisms by which cisplatin and paclitaxel exert their antitumour effects, i.e., cisplatin has a nonspecific effect on the cell cycle, while paclitaxel has a specific effect on the cell cycle [24,25]. Many studies have shown that paclitaxel can cause cancer cell death by regulating apoptosis. In addition to causing mitotic cell cycle arrest, paclitaxel can also induce and activate the transcription of various apoptosis-related genes, thereby inducing apoptosis. Our results ( Figure 5) also suggest that paclitaxel can kill tumor cells by regulating the expression of IMPA2/AIFM2, and this mechanism provides a new target for the major role of paclitaxel as an anti-cervical cancer drug in cancer chemotherapy.
In addition, the potent efficacy of p53 in tumor suppression and the high frequency of p53 variants in cancer have stimulated the development of many cancer therapies targeting the p53 signaling network. For example, the success of retinoic acid versus arsenic therapy for acute promyelocytic leukemia (APL) depends on p53mediated cellular senescence. Although we have revealed many aspects of the p53 signaling network, there is no clear understanding of how p53 performs its multiple functions under certain circumstances, and therefore, it still needs to be studied in detail to improve our understanding of gene regulation mechanisms and nature′s means of defense against cancer. In this study, we found that in addition to the change in IMPA2/AIFM2 expression in paclitaxel-treated cells, the expression of p53 also increased with increasing drug concentration, suggesting that p53 is also involved in regulating paclitaxel-induced apoptosis in cervical cancer cells, i. e., IMPA2/AIFM2/p53 may be a new molecular mechanism for the development of paclitaxel resistance and a potential new target for paclitaxel sensitization therapy.
In conclusion, we identified that IMPA2 knockdown promotes cervical cancer cell apoptosis, and the mechanistic investigation revealed that IMPA2 knockdown triggers AIFM2 expression and activates p53, thereby inducing cancer cell apoptosis. Taken together, our study provides a potential target for cervical cancer therapy. Supplementary data is available at Acta Biochimica et Biophysica Sinica online.